Isolation and properties of malic dehydrogenase from ox-heart mitochondria

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Abstract
A method of purifying malic dehydrogenase from an acetone-dried powder of ox-heart mitochondria is described. A 50-fold purification was achieved by charcoal treatment of an aqueous extract of acetone-dried powder, followed by ammonium sulfate fractionation, ethanol fractionation and electrophoresis in starch. The purified enzyme was electrophoretically homogeneous but diffusion analysis and centrifuging showed the presence of an impurity, in an amount probably less than 10%. The molecular weight was estimated to be 15,000-20,000. Thus malic dehydrogenase is the smallest pyridine nucleotide dehydrogenase known. Evidence is presented for the view that malic dehydrogenase is an [alpha]-hydroxydicarboxylic acid dehydrogenase, active with acids containing 3 to 5 carbon atoms and possessing the same stereochemical arragement about the [alpha]-carbon atom as L-malate. Anomalies in the behavior of the enzyme in relation to the Michaelis-Menten theory are explained by the assumption that the enzyme has 2 sites for the substrate, one of which is catalytically active, the other activating or inhibiting the enzyme, depending on the substrate employed.